Zymogen activation is a process that is not well understood. To increase the understanding of the requirements for trypsinogen activation, we are using the modeling programs of the Computer Graphics Laboratory of the University of California, San Francisco to design into trypsin a metal binding site that is expected induce activation in the presence of certain transition metals. We are redesigning rat anionic trypsin to exploit the spatial relationship of residues Asp-194 and Ile-16 in catalytically active trypsin. In mature trypsin, Asp-194 rotates about the backbone to form a salt bridge with the N-terminal amine of Ile-16. Without this interaction, trypsin is inactive. By substituting amino acid residues in this region with those capable of metal coordination, we expect that, in the presence of metal, the substituted residues will coordinate metal, interacting in a manner similar to that of the salt bridge, and induce activation. We will also model other potential metal binding sites using the modeling software available through the CGL. We will then assay the variant trypsin/trypsinogen proteins for ester and peptide hydrolysis activities, metal presence and affinity, metal binding preference, and kinetically characterize the variants to determine the effects, if any, on specific steps in the catalytic mechanism of trypsin. This investigation will provide valuable information as to the specific molecular requirements for trypsinogen activation and will lay a foundation for the investigation of other zymogens.